CD26 DPP IV as a Drug Design Target

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CD26 (DPP IV) as a Drug Design Target
Department of Biochemistry Tufts
University School of Medicine
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Costimulation through CD26
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GLP-17-36 is NOT Degraded from theAmino Terminus
in CD26-/- Mice
CD26/
CD26-/-
Marguet, D. et al. (2000) PNAS 97, 6874.
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increases insulin secretion
?-cells
glucose
insulin
GLP-1 GIP
Rapidly inactivated by dipeptidyl peptidase IV
meal
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Glucagon-like peptide 1(GLP-1)
A physiological gastrointestinal hormone released
from the lower small intestine
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Glucagon-like peptide-1 (Glp-1)

• Biological Activities
• Stimulation of glucose dependent insulin
  secretion
• Stimulation of insulin biosynthesis
• Inhibition of glucagon secretion
• Inhibition of gastric emptying
• Inhibition of food intake
• May stimulate islet proliferation and islet cell
  neogenesis

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Rapid Degradation by DPP IV

• 2 min after intravenous admin.
• 30 min after subcutaneous admin.
• Production expensive

Cut by DPP IV
dipeptide antagonist
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Pharmacological or genetic inactivation of DP
IV potentiates incretin action and glucose in vivo
DP IV inhibitors are in clinical trials for
treatment of diabetes
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Administration of DPP-IV Inhibitor (TP8211) to
WildType (WT) and Double Incretin Receptor
Knockout (DKO) Mice
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20
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Blood Glucose (mM)
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5
0
0
20
40
60
80
100
120
Time (minutes)
Inhibitor was administered orally (1.25mg/kg) 1
hour prior to oral glucose challenge.
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Xaa-boroPros
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Selectivity Issues with DPP IV Inhibitors

• Select DPP IV inhibitors have demonstrated
  efficacy in animal models of diabetes, but also
  result in toxicities
• It has been proposed that the toxic effects
  associated with these inhibitors arises from the
  inhibition of DPP8 and/or DPP9, and not DPP IV
• A recent study demonstrated that a DPP 8/9
  selective inhibitor was toxic, while a DPP IV
  selective inhibitor was not
• DPP IV-deficient animals present with a normal
  phenotype
• Other DPP IV activity and/or structure homologue
  (DASH) proteins exist whose functions remain
  unknown

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Prolyl Peptidases

• Serine protease subfamily
• Examples of prolyl peptidases
• Functions of many of these proteins unknown

J.S. Rosenblum and Kozarich, J.W. (2003) Curr.
Opin. Chem. Biol. 7, 496.
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Ultra-smarts Or Pro-Soft Drugs
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• pH dependent conformation behavior of
  Xaa-boroPros.
• open chain form at low pH
• cyclic form favored at high pH
• cyclic form inactive

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Tetrapeptide Ultra-smart Prodrug Strategy
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Tetrapeptide Ultra-smart Prodrug Strategy
DPP IV
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DPP IV
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Ultra Smart molecule versions of potent DPP IV
inhibitors exhibit increased potency at high pH
and pH independence in in vitro DPP IV
inhibition assays
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TP-3201 out performs TP01in blocking DPP IV
catalytic activity in vivo in mice.
Normal Control
Diabetic Control
TP-3221
TP01
TP-3201
0.05 mg/kg
1hr post-Rx glucose
88.56
100
77.61
41.29
16.42
4hrs post-Rx glucose
69.16
100
68.56
34.13
18.56
0.025 mg/kg
C
1hr Post Rx-Glucose
82.65
100
111.42
47.49
34.70
4hrs Post Rx-Glucose
89.95
100
58.85
39.23
24.40
0.01 mg/kg
1hr Post Rx-Glucose
82.65
100.00
100.00
59.36
48.86
4hrs Post Rx-Glucose
89.95
100.00
77.51
58.85
40.67
0.0025 mg/kg
1hr Post Rx-Glucose
82.65
100.00
109.59
94.52
84.47
4hrs Post Rx-Glucose
89.95
100.00
99.52
93.30
81.82
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TP-3201 outperforms TP-01 (i.e., more potent,
longer acting, and safer) in vivo in rats. Each
bar represents the average of four animals
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TP-3201 performs very well in oral glucose
tolerance tests in db/db mice at a single oral
dose of 0.05 mg/kg
Test articles given three hours before oral
glucose challenge at T 0 on above graph. Each
time point represents the average of five mice.
Animal work done by MDS Pharma Services,
Montreal, Canada. The data of Fig 2 are these
mice. Note that better than 80 inhibition of
serum enzyme is needed to produce desired effect
on OGTT.
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Smart DPP IV Inhibitor vs GLP-1 in lowering
fasting glucose in db/db mice.
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Smart DPP IV inhibitor vs Exendin-4 in lowering
fasting glucose in mice.

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The greater the hyperglycemia the greater is the
effect exendin-4 exerts in lowering fasting blood
glucose. This shows that TP 3201 does about what
the highest dose of exendin-4 could be expected
to do in the db/db mice to which TP 3201 was
given.
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Val-boroPro Toxicity

• Val-boroPro is not selective for DPP IV
• Val-boroPro is toxic in both wild type and DPP
  IV-deficient rats

Table adapted from results in Lankas, G.R. et al.
(2005) Diabetes 54, 2988.
0.5 mg/kg Val-boroPro
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Chg-Pro-Val-boroPro Reduces Toxicity in Rats
versus Val-boroPro
Pro-soft drug increases specificity for DPP IV,
thus reducing the incidence of interactions of
Val-boroPro with other unintended targets
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Better Targets and Uses for Pro-Soft Drug
Technology
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Ultra-smart Platform Technology S-Xaa-Xaa-T
S Address. Can be target or
tissue specific Xaa-Xaa-T Target
recognition
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New Ultra-Smart Chemical Entities
S substrate for cancer or tissue specific
activator protease. e.g. FAP, PSA,
etc. Xaa-Xaa For enzyme targeted for
inhibition. T boronic acid
alpha keto amide nitrile
trifluoromethylketone aldehyde
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Millennium man Scientist's belief kept study of
cancer-fighting drug alive By Naomi Aoki, Globe
Staff, 12/12/2001

• CAMBRIDGE -- For years, Julian Adams had labored
  on a drug known as LDP-341. He had watched it rid
  mice and monkeys of all signs of cancer without
  the devastating side effects of conventional
  treatments.
• He believed it could be a breakthrough drug, one
  that mounted an entirely new type of attack on
  the deadly disease. But convincing the outside
  world had proved difficult especially as the
  company he founded changed hands twice in a span
  of six months. For a time, he feared his could be
  one of the promising drugs that slipped through
  the cracks in the system.
• Then, in August 2000, came a moment Adams will
  never forget. In an early-stage clinical test,
  the drug erased all signs of cancer from a
  42-year-old woman who months before was in the
  advanced stages of multiple myeloma, an often
  fatal blood-borne form of cancer.

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FDA Approves VELCADE (bortezomib) for Injection
for the Treatment of Relapsed and Refractory
Multiple Myeloma Completely new approach to
treating cancer is the first FDA approved
proteasome inhibitor Cambridge, Mass., May 13,
2003 - Millennium Pharmaceuticals, Inc. (Nasdaq
MLNM) today received approval from the U.S. Food
and Drug Administration (FDA) to market VELCADE
for the treatment of multiple myeloma patients
who have received at least two prior therapies
and have demonstrated disease progression on the
last therapy. The effectiveness of VELCADE is
based on response rates. There are no controlled
trials demonstrating a clinical benefit such as
an improvement in survival. VELCADE, the first of
a new class of medicines called proteasome
inhibitors, is the first treatment in more than a
decade to be approved for patients with multiple
myeloma - a cancer of the blood. "The FDA
approval of VELCADE represents a major advance in
our fight against multiple myeloma," said Ken
Anderson, M.D., director of the Jerome Lipper
Multiple Myeloma Center at Dana-Farber Cancer
Institute in Boston, Mass. and the lead
investigator in the preclinical development and
clinical trials of VELCADE. "With its new and
unique mechanism of action of inhibiting the
proteasome, VELCADE is different from traditional
chemotherapies and represents a new treatment
option for patients."
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The 26S Proteasome

• Found in virtually all living cells
• Comprised of a 20S catalytic and two 19S
  regulatory complexes
• Possesses three distinct types of proteolytic
  activity
• Chymotrypsin-like (rate-limiting)
• Trypsin-like
• Post-glutamyl peptide hydrolyzing

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Proteasome Inhibition as a Treatment for Cancer

• Velcade received accelerated FDA approval on May
  13, 2003 as a treatment for multiple myeloma, a
  cancer of the bone marrow
• Structure of Velcade
• From the FDA Center for Drug Evaluation and
  Research Questions and Answers on Velcade site

Are proteasomes found in only cancer cells? No,
proteasomes are found in all cells, and are
necessary for cells to survive and grow. Velcade
may kill some good cells along with the cancer
cells, which can lead to side effects.
(http//www.fda.gov/cder/drug/infopage/velcade/vel
cadeQA.htm)
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14CVelcade Distribution in Murine Organs and
PC-3 Tumor Tissue
Adams, J. et al. (1999) Cancer Res. 59, 2615.
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O
N
B
O
N
Velcade


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Ultra Smart Velcade

CBZ-CHG-Pro-Phe-Leu-B(OH)2

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Ultra smart Velcade 1. Do NH2Xaa-boroLeu
compds. inhibit? Yes 2. Do
NH2Xaa-boroLeu compds. Cyclize?
Yes 3. Do NH2Xaa-boroLeu compds kill cells?
Yes 4. Can prodrugs of
Xaa-boroPro be made that do not inhibit the
proteasome proteases and do not kill cells.
Yes 5. Can the compds. of 4 above be
activated to inhibit the proteasome protease and
to become lethal to cell? Yes
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PheboroLeu vs. Velcade?
In Vitro 20S Proteasome Inhibition Assay
Cell Toxicity Assay
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Increased Dipeptide Hydrophobicity Correlates
with Improved Toxicity in a Cellular Model
AA hydrophobicity Phe gt Leu gt Gly gt Ser gt His
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Alternate Xaa-boroLeu Dipeptides
Degree of cyclization varies between Xaa-boroLeu
dipeptides
Fold difference between pH 2 and pH 7.6 IC50
values for PheboroLeu 3
IC50 pH 2 21 nM IC50 pH 7.6 461 nM Fold
difference 22
IC50 pH 2 406 nM IC50 pH 7.6 4.4 mM Fold
difference 11
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Fibroblast Activation Protein

• Type II transmembrane prolyl oligopeptidase
• Shares approximately 50 sequence identity with
  DPP IV
• Precise in vivo function is unknown at this time
• Possesses a unique expression profile
• Expressed by soft tissue sarcomas
• Strongly expressed by reactive tumor stromal
  fibroblasts surrounding newly formed blood
  vessels of gt90 of epithelial cancers
• Also expressed in granulation tissue of healing
  wounds and in cirrhotic human liver cells

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Immunoperoxidase Staining of Tumor Tissues with
mAb F19
Rettig, W.J. et al. (1988) PNAS 85, 3110.
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Comparison of Normal vs. Tumor Tissue
Corresponding normal tissue is FAP negative
Given its unique expression profile, FAP
activation of a cancer targeted drug could result
in a more potent and specific drug
Park, J.E. et al. (1999) J. Biol. Chem. 274,
36505.
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Known Enzyme Specificity of FAP
AlaPro-AFC Substrate
DPP Assay
Park, J.E. et al. (1999) J. Biol. Chem. 274,
36505.
When tested against the native ECM proteins
fibronectin, laminin and collagens I and IV,
specific degradation of type I collagen was
observed
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Peptide Libraries as Tools for Assessing Enzyme
Specificity

• Peptide libraries
• Acetylated and non-acetylated hexapeptides
• Substitute one position with all natural amino
  acids with the exception of Cys
• Enzyme specificity pockets

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Mass Spectrometry-based Enzyme Specificity Assay

• Incubate enzyme and peptide library
• at 37C for 24 hrs
• Quench reaction with 1.2 N HCl

• Analyze samples by LC/MS
• Determine the amount of remaining
• intact peptide compared to the library
• alone

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P1 Specificity of FAP
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FAP vs. DPP IV P2 Position
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FAP vs. DPP IV P1 Position
The S1 subsite in FAPa is flat and could
accommodate most amino acids
Aertgeerts, K. et al. (2005) J. Biol. Chem. 280,
19441.
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Cancer TargetedPro-Soft Drug Mechanism
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Rationale for the Design ofSuc-Gly-Pro-Phe-boroLe
u
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Rationale for the Design ofSuc-Gly-Pro-Phe-boroLe
u
HEK293 cells
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Cytotoxicity Results in NIH/3T3 Cells
1406
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